WO2017057523A1

WO2017057523A1 - Astrocyte-like cells and method for preparing same

Info

Publication number: WO2017057523A1
Application number: PCT/JP2016/078741
Authority: WO
Inventors: 淳神山; 智周; 岡野　栄之
Original assignee: 学校法人慶應義塾
Priority date: 2015-09-29
Filing date: 2016-09-28
Publication date: 2017-04-06
Also published as: JPWO2017057523A1; JP6906231B2

Abstract

The present invention pertains to a method for preparing astrocyte-like cells from human cells in vitro, wherein the preparation method includes the following steps: (1) a step for introducing at least two inducers into human cells; and (2) a step for inducing differentiation of the human cells and obtaining astrocyte-like cells. The inducers include NFIA and at least one of NICD and Tet2CD, and the human cells are human somatic cells (excluding astrocytes), human pluripotent stem cells, or human adult stem cells.

Description

Astrocyte-like cells and preparation method thereof

The present invention relates to a method for preparing in vitro astrocyte-like cells from human cells, an astrocyte-like cell prepared by the method, an in vitro neural tissue model including astrocyte-like cells and neurons, and a patient with a nervous system disease The present invention relates to a method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease using an astrocyte-like cell, a human cell-to-astrocyte-like cell inducer, and a human cell-to-astrocyte-like cell induction kit.

Astrocytes are nervous system cells that are gradually being recognized to play a major role in the pathophysiology of many neurological diseases. (Non-Patent Document 1).

In order to study the pathophysiological role of astrocytes for nervous system diseases, a rapid and massive supply of human astrocytes is required. However, at present, the source of human astrocytes is limited. It is ethically impossible to obtain astrocytes by biopsy from a patient with a living nervous system disease. Although it is possible to obtain astrocytes from the postmortem brain of patients with nervous system diseases, mass supply is extremely difficult.

A method for preparing astrocytes by inducing differentiation from human induced pluripotent stem cells (iPS cells) has been reported (Non-patent Document 2).

In addition, NFIA (Nuclear factor I / A), NFIB (Nuclear factor I / B), and SOX9 (SRY (sex determining region Y) -box 9) are introduced into mouse fibroblasts. There has been reported a method of directly inducing (Non-patent Document 3). In the literature, attempts have been made to introduce the three factors into human fibroblasts and induce them into astrocyte-like cells.

It has been reported that the reprogramming efficiency from human fibroblasts to iPS cells can be improved by knocking down the p14ARF gene and the p16INK4 gene or the p16INK4 gene present in the CDKN2A locus (Non-Patent Literature). 4). Moreover, it has been reported that the efficiency of direct reprogramming from human fibroblasts to neurons can be improved by knocking down the p14ARF gene or the p16INK4 gene present in the CDKN2A locus (Non-patent Document 5). ).

P14ARF and p16INK4 are molecules present in different signal transduction pathways, but both signal transduction pathways are involved in cell apoptosis and senescence. MDM2, p53 and p21 are present downstream of p14ARF in the first signal transduction pathway. Further, cyclin D1, CDK4, CDK6, Rb, and E2F exist downstream of p16INK4 in the second signal transduction pathway.

Human fibroblasts by knocking down the p53 gene present downstream of p14ARF in the first signal transduction pathway or by introducing a p53 dominant negative mutant that inhibits the function of the p53 protein into human fibroblasts. It has been reported that the efficiency of reprogramming from cells to iPS cells can be improved (Non-Patent Document 6). It has also been reported that the efficiency of direct reprogramming from human fibroblasts to neurons can be improved by introducing a p53 dominant negative mutant into cells (Non-patent Document 7).

When preparing astrocytes by inducing differentiation from iPS cells, a period of several months is required, and the demand for rapid supply of human astrocytes cannot be satisfied.

In addition, in the method reported in Non-Patent Document 3, after introducing NFIA, NFIB and SOX9 into human neonatal fibroblasts, only 2% of astrocyte-like cells are present after 3 weeks of culture. Not observed, its induction efficiency is very low.

Therefore, the present invention provides a novel method that enables the rapid and highly efficient preparation of astrocyte-like cells having the same characteristics as human astrocytes that are extremely difficult to obtain using easily obtainable human cells. Let it be an issue.

As a result of intensive studies in view of the above problems, the present inventors have determined that human somatic cells have at least one of specific inducers (NICD (Notch 1 intercellular domain) and Tet2CD (TET (Ten-eleven translocation) 2 catalytic domain)), And NFIA), it was found that human astrocyte-like cells can be prepared with high efficiency in a very short period of time without preparation of iPS cells. Furthermore, the present inventors have used human astrocyte-like cells for a very short period of time even when an inducer similar to the above is introduced into pluripotent stem cells such as iPS cells and adult stem cells such as neural stem cells. And found that it can be prepared with high efficiency. The present invention is based on these surprising findings.

That is, this invention has the following aspects.
[1]
A method for preparing in vitro astrocyte-like cells from human cells comprising the following steps:
(1) introducing at least two kinds of inducers into human cells; and (2) obtaining astrocyte-like cells by inducing differentiation of the human cells,
Wherein the inducer comprises NFIA and comprises at least one of NICD and Tet2CD;
The preparation method, wherein the human cells are human somatic cells (excluding astrocytes), human pluripotent stem cells or human adult stem cells.
[2]
The preparation method according to [1], wherein the inducer includes NFIA, NICD, and Tet2CD.
[3]
The preparation method according to [1] or [2], wherein the human cell is a human fibroblast.
[4]
The preparation method according to [1] or [2], wherein the human cell is a human iPS cell or a human neural stem cell.
[5]
The human cell is a cell derived from a patient with a nervous system disease, or a cell derived from a healthy person artificially introduced with a mutation that specifically exists in the gene of a cell derived from the patient with a nervous system disease , [1] to [4].
[6]
The preparation method according to any one of [1] to [5], wherein the introduction of the inducer in the step (1) includes introducing at least one vector containing a nucleic acid encoding the inducer.
[7]
In the step (1),
inhibits the expression of at least one gene selected from p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene,
inhibits at least one selected from p14ARF, p53, p21, p16INK4a and Rb,
Increases the expression of at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, or activates at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F The preparation method according to any one of [1] to [6], further comprising:
[8]
An astrocyte-like cell prepared by the method according to any one of [1] to [7].
[9]
A method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, comprising the following steps:
(1) From a cell derived from a patient with a nervous system disease, or a cell derived from a healthy person into which a mutation that is specifically present in the gene of a cell derived from a patient with the nervous system disease has been artificially introduced into a gene, [1] to [ 7] a step of preparing astrocyte-like cells by the method according to any one of the above; and (2) contacting the test substance with the astrocyte-like cells obtained in step (1), Said method comprising the step of assessing efficacy or toxicity.
[10]
A method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, comprising the following steps:
(1) A step of preparing astrocyte-like cells from cells derived from healthy subjects by the method according to any one of [1] to [7];
(2) a step of knocking out or knocking down a nervous system disease-related gene in the astrocyte-like cell obtained in step (1); and (3) an astrocyte-like cell obtained in step (2) and a test substance The method comprising the steps of: contacting and evaluating the efficacy or toxicity of the test substance.
[11]
An in vitro neural tissue model comprising the astrocyte-like cell according to [8] and a neuron.
[12]
A method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, comprising the following steps:
(1) From a cell derived from a patient with a nervous system disease, or a cell derived from a healthy person into which a mutation that is specifically present in the gene of a cell derived from a patient with the nervous system disease has been artificially introduced into a gene, [1] to [ 7] a step of preparing astrocyte-like cells by the method according to any one of
(2) a step of preparing a neural tissue model by co-culturing astrocyte-like cells and neurons obtained in step (1);
(3) The said method including the process of contacting the nerve tissue model obtained by process (2), and a test substance, and evaluating the effectiveness or toxicity of a test substance.
[13]
A method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, comprising the following steps:
(1) A step of preparing astrocyte-like cells from cells derived from healthy subjects by the method according to any one of [1] to [7];
(2) a step of knocking out or knocking down a nervous system disease-related gene in the astrocyte-like cell obtained in step (1);
(3) a step of preparing a neural tissue model by co-culturing astrocyte-like cells and neurons obtained in step (2);
(4) The method comprising the step of contacting the nerve tissue model obtained in step (3) with a test substance to evaluate the effectiveness or toxicity of the test substance.
[14]
An inducer from a human cell to an astrocyte-like cell, comprising at least one kind of inducer or at least one vector containing a nucleic acid encoding the inducer, wherein the inducer is NFIA And at least one of NICD and Tet2CD, and the human cell is a human somatic cell (excluding astrocytes), a human pluripotent stem cell or a human adult stem cell.
[15]
Including at least two types of inducers, or at least one vector containing a nucleic acid encoding the inducer; and an instruction manual describing the method according to any one of [1] to [7], A kit for inducing an astrocyte-like cell from a human cell, wherein the inducer includes NFIA and includes at least one of NICD and Tet2CD, and the human cell is a human somatic cell (an astrocyte). Except), which is a human pluripotent stem cell or a human adult stem cell.
[16]
an expression inhibitor of at least one gene selected from p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and / or at least one inhibitor selected from p14ARF, p53, p21, p16INK4a and Rb, and And / or expression enhancer of at least one gene selected from MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, and / or at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F The kit according to [15], further comprising an activator.

According to the method of the present invention, it becomes possible to supply human astrocytes quickly and in large quantities. In addition, it is possible to construct a new in vitro disease model using human astrocytes obtained by the method of the present invention.

Induction after introducing a lentiviral vector containing a nucleic acid encoding NFIA, a lentiviral vector containing a nucleic acid encoding NICD, and a lentiviral vector containing a nucleic acid encoding Tet2CD into human fibroblasts and culturing for 11 days The percentage of GFAP-expressing cells made is shown. The vertical axis represents the ratio of the number of GFAP positive cells to the number of DAPI positive cells. In the figure, “Fibro” indicates human fibroblasts into which no inducer has been introduced. “NFIA”, “Tet2CD”, and “NICD” indicate human fibroblasts introduced with NFIA, Tet2CD, and NICD, respectively. In the figure, “NFIA / Tet2CD”, “Tet2CD / NICD”, and “NICD / NFIA” are human fibroblasts introduced with NFIA and Tet2CD, human fibroblasts introduced with Tet2CD and NICD, NICD and NFIA, respectively. Shows human fibroblasts introduced with. In the figure, “NIFA / NICD / Tet2CD” indicates human fibroblasts into which NFIA, NICD and Tet2CD have been introduced. After co-culture of astrocyte-like cells with neurons, an image of the neurons and a measurement result of the length of neurites extended from the neurons by a fluorescence microscope are shown. The scale bar in the image is 100 μm. Fluorescence microscope images were obtained by labeling neurons with anti-MAP2 antibody (Sigma). The vertical axis of the graph represents the average neurite length per neuron cell. In the figure, “w / o” indicates a case where the cells are not co-cultured with astrocyte-like cells (that is, only neurons). In the figure, “w / NHDF-Ad” indicates a case where human adult dermal fibroblasts (NHDF-Ad) and neurons are co-cultured. In the figure, “w / NHDF-Neo” indicates the case where human neonatal skin fibroblasts (NHDF-Neo) and neurons are co-cultured. In the figure, “w / iAs (NHDF-Ad)” indicates a case where astrocyte-like cells derived from human adult dermal fibroblasts and neurons are co-cultured. In the figure, “w / iAs (NHDF-Neo)” indicates a case where astrocyte-like cells derived from human neonatal dermal fibroblasts and neurons are co-cultured. In the figure, “w / hAstrocytes” indicates a case where human astrocytes and neurons are co-cultured. The measurement result of the glutamate uptake | capture ability of an astrocyte-like cell is shown. A retroviral vector containing a nucleic acid encoding NFIA, a retroviral vector containing a nucleic acid encoding NICD, and a retroviral vector containing a nucleic acid encoding Tet2CD are human lt-NES cells (long-term self-renewing neuroepithelial stem cells) The ratio of the induced GFAP-expressing cells after introduction into 1 and culturing for 1 week and 2 weeks is shown. The vertical axis shows the ratio of the number of GFAP positive cells to the total number of cells. In the figure, “lt-NES iA” indicates astrocyte-like cells derived from lt-NES cells. Figure 2 shows fluorescence microscopic images of astrocyte-like cells derived from human adult fibroblasts and lt-NES cells using lentiviral vectors containing all of the nucleic acids encoding NFIA, NICD, and Tet2CD. FIG. 6 shows that the expression level of CRYAB was increased in an in vitro model of Alexander disease obtained by inducing differentiation from lt-NES cells as compared to the control group. The vertical axis represents the ratio of the number of GFAP-positive and CRYAB (Alpha-crystallin B) positive cells to the number of GFAP-positive cells. In the figure, “Control” represents an astrocyte-like cell derived from an lt-NES cell into which no missense point mutation (C715T) has been introduced, and “AxD” represents an lt into which a missense point mutation (C715T) has been introduced. -Shows astrocyte-like cells derived from NES cells. When a lentiviral vector expressing a short hairpin RNA (shRNA) against MeCP2 is introduced into fibroblast-derived astrocyte-like cells and co-cultured with mouse E15.5 primary cultured cerebral cortical neurons, the neurons are examined by fluorescence microscopy. An image and the measurement result of the length of the neurite extended from the neuron are shown. Fluorescence microscope images were obtained by labeling neurons with anti-MAP2 antibody (Sigma). In the figure, “w / iA (control)” shows the result of a coculture of astrocyte-like cells not expressing shRNA against MECP2 and neurons (control group). In the figure, “w / iA (shMeCP2)” shows the result of co-culturing astrocyte-like cells and neurons expressing shRNA against MECP2. In the figure, “w / iA (Rescue)” indicates a coculture of astrocyte-like cells and neurons expressing both shRNA against MECP2 and wild-type MECP2 whose expression is not suppressed by shRNA. The vertical axis of the graph represents the ratio of the average neurite length in the co-culture group to the average neurite length per neuron in the control group. Measurement of length of neurites extended from neurons when lentiviral vector expressing shRNA against MeCP2 is introduced into astrocyte-like cells derived from lt-NES cells and cultured with mouse E15.5 primary cultured cerebral cortical neurons Results are shown. The vertical axis represents the average neurite length per neuron cell. In the figure, “w / iA (shMeCP2)” shows the result of co-culture of neurons and astrocyte-like cells expressing shRNA against MECP2. In the figure, “w / iA (Rescue)” indicates a co-cultured astrocyte-like cell and neuron expressing both shRNA against MECP2 and mutant MECP2 not suppressed by shRNA. The result at the time of introduce | transducing p16INK4a into a cell in the differentiation induction from a human fibroblast to an astrocyte-like cell is shown. In the figure, “3 factor” is a human fibroblast introduced with a lentiviral vector containing a nucleic acid encoding NFIA, a lentiviral vector containing a nucleic acid encoding NICD, and a lentiviral vector containing a nucleic acid encoding Tet2CD. Indicates that there is. In the figure, “+ p16INK4a” indicates the result when a lentiviral vector containing a nucleic acid encoding p16INK4a is introduced into a cell in addition to the above-described three-encoding vector. In the figure, “+ Ctl” indicates the result when an empty lentiviral vector not containing a gene sequence to be expressed is used instead of a lentiviral vector containing a nucleic acid encoding p16INK4a. The vertical axis (% 左 of GFAP (+) / DAPI) in the left bar graph shows the ratio of the number of GFAP positive cells to the number of DAPI positive cells after culturing for 11 days after introducing the vector encoding the inducer into the cells (GFAP (Number of positive cells / number of DAPI positive cells) is expressed in%. The vertical axis (% 棒 of GFAP (+) / Initial cells) on the right bar graph shows the number of GFAP-positive cells collected after 11 days of introduction of the vector encoding the inducer into the cells and at the start of culture. The percentage of the initial cell number (GFAP positive cell number / initial cell number) is shown in%. The result at the time of knocking down the intracellular p16INK4a gene in the differentiation induction from a human fibroblast to an astrocyte-like cell is shown. In the figure, the definitions of “3 factors”, “% of GFAP (+) / DAPI” and “% of GFAP (+) / DAPI” are the same as those in FIG. In the figure, “+ shp16INK4a” indicates the result when a lentiviral vector containing a nucleic acid encoding a short hairpin RNA (shp16INK4a) for the p16INK4a gene is introduced into the cells in addition to the vector encoding the above three factors. In the figure, “+ Ctl” indicates that a lentiviral vector containing a nucleic acid encoding a short hairpin RNA targeting a sequence that does not exist in humans is introduced into a cell instead of a lentiviral vector containing a nucleic acid encoding shp16INK4a. The result is shown below. The result when the intracellular p53 gene is knocked down in the induction of differentiation from human fibroblasts to astrocyte-like cells is shown. In the figure, the definitions of “3 factors”, “% of GFAP (+) / DAPI” and “% of GFAP (+) / DAPI” are the same as those in FIG. In the figure, “+ shp53” indicates the result when a lentiviral vector containing a nucleic acid encoding a short hairpin RNA (shp53) for the p53 gene in addition to the three factors is introduced into the cell. In the figure, “+ Ctl” indicates that a lentiviral vector containing a nucleic acid encoding a short hairpin RNA whose target sequence is a non-human sequence is introduced into a cell instead of a lentiviral vector containing a nucleic acid encoding shp53. The result is shown below. The result at the time of knocking down the intracellular p14ARF gene in the differentiation induction from a human fibroblast to an astrocyte-like cell is shown. In the figure, the definitions of “3 factors”, “% of GFAP (+) / DAPI” and “% of GFAP (+) / DAPI” are the same as those in FIG. In the figure, “+ shp14ARF” indicates the result when a lentiviral vector containing a nucleic acid encoding a short hairpin RNA (shp14ARF) for the p14ARF gene in addition to the three factors is introduced into the cell. In the figure, “+ Ctl” indicates a case where a lentiviral vector containing a nucleic acid encoding a short hairpin RNA whose target sequence is a non-human sequence is introduced into a cell instead of a vector containing a nucleic acid encoding shp14ARF The results are shown. The results when knocking down the intracellular p21 gene in the induction of differentiation from human fibroblasts to astrocyte-like cells are shown. In the figure, the definitions of “3 factors”, “% of GFAP (+) / DAPI” and “% of GFAP (+) / DAPI” are the same as those in FIG. In the figure, “+ shp21” indicates the result when a lentiviral vector containing a nucleic acid encoding a short hairpin RNA (shp21) for the p21 gene in addition to the three factors is introduced into the cell. In the figure, “+ Ctl” indicates a case where a lentiviral vector containing a nucleic acid encoding a short hairpin RNA whose target sequence is a non-human sequence is introduced into a cell instead of a vector containing a nucleic acid encoding shp21 The results are shown. Percentage of GFAP-expressing cells induced after introducing one, two or three of mouse NICD, mouse Tet2CD, mouse NFIA, and mouse REST / NRSF-VP16 into mouse fibroblasts and culturing. Show. The vertical axis represents the number of GFAP-expressing cells per cm ² . The abscissa indicates the introduced inducer.

One aspect of the present invention is a method for preparing in vitro astrocyte-like cells from human cells, the following steps: (1) introducing at least two types of inducers into human cells; and (2) Inducing differentiation of a human cell to obtain an astrocyte-like cell, wherein the inducer includes NFIA and includes at least one of NICD and Tet2CD, and the human cell is a human somatic cell (astrocytoma). The above preparation method is a human pluripotent stem cell or a human adult stem cell. Hereinafter, the preparation method is also referred to as “the preparation method of the astrocyte-like cell of the present invention” or “the preparation method of the present invention”.

In the present specification, “astrocyte-like cells” are cells obtained by inducing differentiation of human somatic cells (excluding astrocytes), human pluripotent stem cells or human adult stem cells in vitro, A cell that has the same characteristics as a site. Specifically, it has the following characteristics.
(1) It expresses glial fibrillary acidic protein (GFAP), which is a specific marker for astrocytes.
(2) Ability to support neurite outgrowth from neurons.
(3) It has the ability to take in glutamic acid.
In the present specification, the astrocyte-like cell is also referred to as “induced astrocyte” or “iA cell”.

As used herein, “inducing factor” refers to a protein that can induce differentiation of human somatic cells (excluding astrocytes), human pluripotent stem cells, and adult human stem cells into astrocyte-like cells. The inducer used in the method for preparing an astrocyte-like cell of the present invention contains NFIA (Nuclear factor I / A) and includes at least one of NICD (Notch-1 intercellular domain) and Tet2CD (Tet2 catalytic domain). . Specifically, the inducer may include NFIA and NICD, may include NFIA and Tet2CD, and may include NFIA, NICD and Tet2CD. Preferably, the inducer comprises NFIA, NICD and Tet2CD. In addition to the wild type protein, the inducer has 90% or more, preferably 95% or more, more preferably 98% or more, particularly preferably 99% or more identity with the amino acid sequence of the wild type protein. It may be a mutant protein having the same ability to induce astrocyte-like cells as the protein. For example, when mouse NFIA, mouse NICD and mouse Tet2CD are selected as inducers, not only their wild-type proteins (SEQ ID NO: 8, 9 and 10 respectively) but also their sequences and 90% or more, preferably 95 %, More preferably 98% or more, particularly preferably 99% or more, and the use of a mutant protein having the ability to induce astrocytic cells is also used in the preparation method of the present invention. include. In addition to the inducer protein itself, the inducer may be in the form of a fusion protein of the protein and another protein, polypeptide or peptide. The other protein, polypeptide or peptide is not particularly limited, but protein membranes such as His tag, FLAG tag, Myc tag, V5 tag, PA tag and HA tag, and cell membrane such as TAT peptide derived from HIV virus Contains a permeable peptide. As long as the fusion protein has the ability to induce astrocytic cells, the use of the fusion protein is also included in the use of the inducer in the preparation method of the present invention.

The origin of the inducer used in the preparation method of the present invention is not particularly limited, but is preferably derived from a mammal, more preferably a primate (human, monkey, etc.), rodent (mouse, rat, guinea pig, etc.). ), Cats, dogs, rabbits, sheep, pigs, cows, horses, donkeys, goats or ferrets, particularly preferably mice or humans. The combination of inducers used in the preparation method of the present invention may be a combination of inducers derived from different species or a combination of inducers derived from the same species. Preferably, the combination of inducers used in the preparation method of the present invention is a combination of inducers derived from the same species.

As used herein, the term “somatic cell” refers to a germline cell (eg, egg cell, sperm cell, oocyte cell, spermatogonia cell, etc. and their precursor cells) or a universal undifferentiated cell derived from an early embryo ( For example, it is a differentiated cell constituting an animal individual other than embryonic stem cells). The animal individual may be an adult, a fetus or an embryo. The somatic cell may be an established cell or a primary cultured cell isolated from a tissue, but preferably has a normal number of chromosomes. Tissues and organs from which somatic cells are derived are not particularly limited, and include skin, liver, blood and the like. The characteristics of somatic cells are not particularly limited as long as they are cells that have lost all of the differentiation potential of fertilized cells, and include, for example, fibroblasts, epithelial cells, hepatocytes, peripheral blood cells and the like.

In the method for preparing an astrocyte-like cell of the present invention, the human somatic cell used as a starting cell is not particularly limited as long as the astrocyte-like cell is induced to differentiate, but a human fibroblast or a human peripheral blood cell Is preferred.

In the present specification, “pluripotent stem cells” include all cells having differentiation pluripotency that can be differentiated into all cells other than placenta, and include embryonic stem cells (Embryonic Stem cell (ES cells)) and artificial pluripotent cells. It contains potent stem cells (induced Pluripotent Stem cells (iPS cells)).

“ES cell” refers to a stem cell made from an inner cell mass belonging to a part of an embryo at the blastocyst stage, which is an early stage of animal development.

“IPS cells” are cells that have been reprogrammed (initialized) to have the same pluripotency as ES cells by introducing specific factors into mammalian somatic cells or undifferentiated stem cells. is there.

iPS cells were first established by introducing four factors Oct3 / 4, Sox2, Klf4, and c-Myc into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663- 676). Subsequently, human iPS cells were also established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S. et al., Cell, (2007) 131: 861-872.), And c-Myc It has also succeeded in establishing a method for establishing safer iPS cells with low tumorigenesis, such as methods not included (NakagawaNM, Yamanaka S., Nature Biotechnology, (2008) 26, 101-106).

In addition, induced pluripotent stem cells prepared by introducing different factors (OCT3 / 4, SOX2, NANOG, LIN28) into human fibroblasts have been reported (Yu （J., Thomson JA., Etc.). Science, 2007, 318: 1917-1920.). In addition, an induced pluripotent stem cell prepared by introducing 6 genes of OCT3 / 4, SOX2, KLF4, C-MYC, hTERT, mSV40large T into skin cells has been reported (Park IH, Daley GQ. Et al., Nature , 2007, 451: 141-146). In addition, artificial pluripotent stem cells prepared by introducing OCT3 / 4, KLF4, and low molecular compounds into mouse neural progenitor cells (Shi Y., Ding S. et al., Cell Stem Cell, 2008, Vol3, Issue, 5,568 -574), induced pluripotent stem cells produced by introducing OCT3 / 4, KLF4 into mouse neural stem cells that endogenously express SOX2, C-MYC (Kim JB., Scholer HR. Et al., Nature, (2008) 454,646-650), artificial pluripotent stem cells prepared using Dnmt inhibitors and HDAC inhibitors without using C-MYC (Huangfu D., Melton, DA. Et al., Nature Biotechnology, ( 2008) 26, No 7, 795-797).

In the present specification, an “artificial pluripotent stem cell (iPS cell)” is a known artificial pluripotent stem cell and an equivalent artificial pluripotency as long as the above definition is satisfied and the object of the present invention is not impaired. Including all stem cells, the cell source, introduction factor, introduction method and the like are not particularly limited.

As used herein, “adult stem cells” are non-differentiated cells found in differentiated tissues and have the ability to proliferate and differentiate into one or more cell types. Examples of adult stem cells include neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and the like. As an adult stem cell used in the method for preparing an astrocyte-like cell of the present invention, a neural stem cell is preferable. The origin of the adult stem cell used in the method for preparing an astrocyte-like cell of the present invention is not particularly limited, and may be obtained from a human living body or obtained by induction from a pluripotent stem cell. It may be. As an adult stem cell used in the method for preparing an astrocyte-like cell of the present invention, an adult stem cell derived from a pluripotent stem cell is preferable, and a human neural stem cell derived from a human iPS cell (for example, derived from a human iPS cell). More preferred are lt-NES cells).

In one embodiment of the method for preparing astrocyte-like cells of the present invention, the starting human cell may be a cell derived from a patient with a nervous system disease. Although it does not specifically limit as said nervous system disease, For example, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, autism, Alexander disease, Rett syndrome etc. are mentioned.

In one embodiment of the method for preparing astrocyte-like cells of the present invention, the starting human cell may be a cell derived from a healthy person who does not suffer from a nervous system disease. The cell derived from the healthy subject may be a cell derived from a healthy subject into which a mutation specifically present in the gene of a cell derived from a patient with a nervous system disease has been introduced. Mutation can be introduced by methods known to those skilled in the art (eg, CRISPR-Cas9 series (Ran, FA et al., Cell, 2013, 154, 1380-1389), genome editing using ZFN (Urnov, F. et al. , Nature, 2005, 435, 646-651), or genetic modification by TALEN (Mahfouz, M et al., PNAS, 2011, 108, 2623-2628)).

In the method for preparing an astrocyte-like cell of the present invention, the method for introducing an inducer into a human cell as a starting cell is not particularly limited. For example, the inducer itself may be directly introduced into a cell (protein introduction method). Alternatively, at least one vector containing a nucleic acid encoding an inducer may be introduced into human cells to express the inducer (gene transfer method).

The method of introducing the inducer into human cells by introducing the protein itself is not particularly limited, and any method known to those skilled in the art may be used. For example, a method using a protein introduction reagent, a method using a protein introduction domain (PTD) or a cell-penetrating peptide (CPP) fusion protein, a microinjection method, an electroporation method and the like can be mentioned.

On the other hand, when a gene introduction method for introducing an inducer into a human cell is used, first, a method well known to those skilled in the art is used, and downstream of an appropriate promoter for expressing the nucleic acid encoding the inducer in a human cell. Make at least one inserted vector. In the present specification, the “nucleic acid encoding an inducer” may be DNA or RNA, or may be a DNA / RNA chimera. The nucleic acid may be double-stranded or single-stranded. Preferably, the nucleic acid is double stranded DNA, in particular cDNA. The nucleic acid encoding the inducer may be a mutated sequence as long as the expressed protein has the same ability to induce astrocyte-like cells as the wild-type inducer. In the present specification, “at least one vector containing a nucleic acid encoding an inducer” may incorporate each of the nucleic acids on a separate vector, or two or more types, preferably 2 to 3 types, in one vector. Of the nucleic acid may be incorporated.

The vector that can be used in the method for preparing an astrocyte-like cell of the present invention is not particularly limited, and for example, a plasmid vector, a virus vector (for example, a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, Sendai virus vector), Artificial chromosome vectors, episomal vectors and the like can be mentioned. The vector used in the method for preparing an astrocyte-like cell of the present invention is preferably a lentiviral vector.

As a method for introducing a vector into human cells, any method known to those skilled in the art, such as an electroporation method, a calcium phosphate method, a lipofection method, or a method via viral infection, may be used. Thus, an inducer can be introduced into a human cell by introducing a vector capable of expressing the inducer into a human cell and expressing the inducer in the human cell.

By culturing human cells into which an inducer has been introduced, differentiation into astrocyte-like cells can be induced. The medium to be used is not particularly limited. For example, Dulbecco's modified Eagle medium (DMEM) or astrocyte growth medium (AGM medium (trademark), Lonza) can be used. The culture temperature is preferably 33 to 37 ° C, more preferably 37 ° C. The culture period is not particularly limited as long as astrocyte-like cells can be collected, but it can be usually collected in about 1 to 2 weeks.

There are signal transduction pathways via p14ARF, MDM2, p53, and p21 as signal transduction pathways involved in cell apoptosis and senescence. Specifically, p14ARF inhibits MDM2. MDM2 also inhibits p53 activation by inhibiting transcriptional regulation by p53, promoting p53 degradation, and promoting p53 nuclear export. P53 activates p21 by transcriptional activation of the p21 gene. P21 promotes apoptosis and senescence of cells by regulating the expression of genes related to apoptosis and senescence.

Further, as another signal transduction pathway involved in cell apoptosis and aging, there is a signal transduction pathway via p16INK4a, cyclin D1, CDK4, CDK6, Rb, and E2F. Specifically, p16INK4a inhibits the activation of cyclin D1, CDK4 and CDK6 by inhibiting the binding between cyclin D1 and CDK4 / 6. The complex of cyclin D1 and CDK4 / 6 inhibits the binding between Rb and E2F by phosphorylating Rb. Since E2F is suppressed by the binding of Rb protein, it has a transcriptional activation effect by phosphorylation of Rb. E2F suppresses apoptosis and senescence of cells by regulating the expression of genes related to apoptosis and senescence.

It has been reported that the efficiency of induction into iPS cells or neurons increases by inhibiting signal transduction starting from p14ARF or p16INK4a in the above two pathways. Surprisingly, it became clear that inhibition of the signal transduction also increased the induction efficiency into astrocyte-like cells.

Therefore, in step (1) of the method for preparing an astrocyte-like cell of the present invention, the expression of at least one gene selected from the p14ARF gene, the p53 gene, the p21 gene, the p16INK4a gene and the Rb gene is inhibited, or p14ARF, inhibits at least one selected from p53, p21, p16INK4a and Rb, or increases the expression of at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, or It may further include activating at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F. Preferably, in step (1) of the method for preparing astrocyte-like cells of the present invention, the expression of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene is inhibited, or p14ARF Inhibiting at least one selected from p53, p21, p16INK4a and Rb or increasing the expression of at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene Further, it may be included. More preferably, in the step (1) of the method for preparing an astrocyte-like cell of the present invention, the expression of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene is inhibited, It may further comprise inhibiting at least one selected from p14ARF, p53, p21, p16INK4a and Rb. More preferably, in the step (1) of the method for preparing an astrocyte-like cell of the present invention, the expression of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene is inhibited. Further, it may be included. Particularly preferably, step (1) of the method for preparing an astrocyte-like cell of the present invention may further comprise inhibiting the expression of at least one gene selected from the p14ARF gene, the p53 gene, the p21 gene and the p16INK4a gene. good.

The method for inhibiting the expression of at least one gene selected from the p14ARF gene, the p53 gene, the p21 gene, the p16INK4a gene and the Rb gene is not particularly limited, and can be performed by any method known to those skilled in the art. For example, it may be carried out by introducing a nucleic acid (for example, one that causes RNAi, such as siRNA, shRNA and miRNA) into a human cell. In addition, knockout by genome editing using CRISPR-Cas9 series (Ran, FA et al., Cell, 2013, 154, 1380-1389), gene modification using ZFN (Urnov, F. et al., Nature, 2005, 435, 646-651 ), Or genetic modification using TALEN (Mahfouz, M et al., PNAS, 2011, 108, 2623-2628)). Inhibition of the expression of the gene is preferably performed using siRNA, shRNA, or miRNA that knocks down the target gene, and more preferably using shRNA.

When inhibition of the expression of the gene is carried out using shRNA, it is not particularly limited, but is preferably encoded by DNA having the base sequence represented by SEQ ID NO: 14 or DNA having the base sequence represented by SEQ ID NO: 15. RNA (shRNA that knocks down the p14ARF gene), DNA having the base sequence represented by SEQ ID NO: 16 or RNA encoded by the DNA having the base sequence represented by SEQ ID NO: 17 (shRNA that knocks down the p53 gene gene), RNA encoded by DNA having the base sequence shown by SEQ ID NO: 18 or DNA having the base sequence shown by SEQ ID NO: 19 (shRNA that knocks down the p21 gene), or has the base sequence shown by SEQ ID NO: 20 DNA or SEQ ID NO Is performed using RNA (shRNA knocking down p16INK4a gene) encoded by DNA having the nucleotide sequence represented by 1.

The method for inhibiting at least one selected from p14ARF, p53, p21, p16INK4a and Rb is not particularly limited, and can be performed by any method known to those skilled in the art. For example, p53 dominant negative mutant that inhibits p53, inactive Rb mutant, pifthrin-α (a transcription inhibitor of p53), and the like may be introduced into human cells.

The method for increasing the expression of at least one gene selected from the MDM2 gene, the cyclin D1 gene, the CDK4 gene, the CDK6 gene and the E2F gene is not particularly limited, and can be performed by any method known to those skilled in the art. For example, these genes can be forcibly expressed in human cells using an expression vector incorporating the above genes, or an inactive ZFN nuclease fused with a transcriptional activator such as VP16, VP64, VP128 (Sanchez, JP, et al., Plant Biotechnol J, 2006, 4 (1), 103-114), inactive TALEN nuclease (Perez-Pinera, P. et al., Nat Methods, 2013, 10, 239-242) or inactive Cas9 It may be performed by increasing the endogenous transcripts of the above genes using nucleases (Konermann, S. et al., Nature, 2015, 517, 583-588).

The method for activating at least one selected from MDM2, cyclin D1, CDK4, CDK6, and E2F is not particularly limited, and can be performed by any method known to those skilled in the art.

The inhibition and increase of the expression of the above gene and the inhibition and activation of the gene product of the gene may be performed simultaneously with the introduction of the inducer into human cells, or may be performed before or after that.

One embodiment of the present invention relates to an astrocyte-like cell prepared by the preparation method of the present invention. One embodiment of the present invention comprises the following steps: (1) introducing at least two types of inducers into human cells; and (2) obtaining astrocyte-like cells by inducing differentiation of the human cells. An astrocyte-like cell prepared by a method comprising: wherein the inducer comprises NFIA and comprises at least one of NICD and Tet2CD, wherein the human cell is a human somatic cell (excluding astrocytes) ), The astrocyte-like cell, which is a human pluripotent stem cell or a human adult stem cell.

For example, when a cell derived from a patient with a nervous system disease is used as a starting cell in the method for preparing an astrocyte-like cell of the present invention, or a mutation that specifically exists in a cell derived from a patient with a nervous system disease is artificially introduced When prepared healthy human cells are used, the prepared astrocyte-like cells retain the genetic characteristics unique to the nervous system disease. Therefore, the astrocyte-like cell can be used as a nervous system disease model for elucidation of the onset cause and pathological progress mechanism of the nervous system disease. The astrocyte-like cells can be used to evaluate the effectiveness or toxicity of a test substance against the nervous system disease, and can be used for drug discovery research.

One aspect of the present invention is a method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, which comprises the following steps: (1) a cell derived from a neurological disease patient or a cell derived from a neurological disease patient A step of preparing an astrocyte-like cell from a cell derived from a healthy subject into which a mutation that is specifically present in is introduced by the method of preparing an astrocyte-like cell of the present invention; and (2) the astrocyte-like cell; The method includes the step of contacting the test substance and evaluating the effectiveness or toxicity of the test substance. Although it does not specifically limit as said nervous system disease, For example, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, autism, Alexander disease, Rett syndrome etc. are mentioned.

In astrocyte-like cells prepared from cells derived from healthy individuals by the preparation method of the present invention, a gene related to a nervous system disease may be knocked out or knocked down. In the present specification, the “neurological disease-related gene” is or is likely to cause a nervous system disease directly or indirectly by the gene not functioning normally due to mutation or the like. There is a gene. Knockout or knockdown can be performed by methods well known to those skilled in the art (for example, knockout by genome editing using CRISPR-Cas9 series (Ran, FA et al., Cell, 2013, 154, 1380-1389), gene modification using ZFN (Urnov, F. et al., Nature, 2005, 435, 646-651), knockout by TALEN genetic modification (Mahfouz, M et al., PNAS, 2011, 108, 2623-2628), siRNA, shRNA, miRNA, etc. (Knockdown by RNAi to be used). The obtained astrocyte-like cells can be used as a nervous system disease model.

One aspect of the present invention is a method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, which comprises the following steps: (1) A method for preparing an astrocyte-like cell of the present invention from a cell derived from a healthy subject (2) a step of knocking out or knocking down a gene related to nervous system disease in the astrocyte-like cell obtained in step (1); and (3) in step (2) The method includes the step of contacting the obtained astrocyte-like cell with a test substance to evaluate the effectiveness or toxicity of the test substance. Although it does not specifically limit as said nervous system disease, For example, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, autism, Alexander disease, Rett syndrome etc. are mentioned.

One embodiment of the present invention relates to an in vitro neural tissue model including astrocyte-like cells and neurons prepared by the preparation method of the present invention. Hereinafter, the neural tissue model is also referred to as “neural tissue model of the present invention”.

The astrocyte-like cell used in the neural tissue model of the present invention may be a cell induced to differentiate from a cell derived from a patient with a nervous system disease, or may be a cell induced to differentiate from a cell derived from a healthy person. Alternatively, it may be a cell derived from a healthy person in which a mutation that specifically exists in the gene of a cell derived from a patient with a nervous system disease is artificially introduced into the gene.

In addition, the astrocyte-like cell used in the neural tissue model of the present invention is a cell that is induced to differentiate from a cell derived from a healthy subject, and may be a cell in which a gene related to a nervous system disease is knocked out or knocked down. Good.

The neuron used in the neural tissue model of the present invention is not particularly limited as long as it is a mammal-derived neuron, and is, for example, a mouse neuron or a human neuron. The neuron may be a neuron derived from a patient with a nervous system disease or a neuron derived from a healthy person, and a mutation specifically present in the gene of a cell derived from a patient with a nervous system disease is introduced into the gene. It may be a neuron derived from a healthy person. The neuron may be a neuron derived from a pluripotent stem cell or an adult stem cell derived from a patient with a nervous system disease, or a neuron derived from a pluripotent stem cell or an adult stem cell derived from a healthy person. Good.

The neural tissue model of the present invention can be obtained by co-culturing astrocyte-like cells and neurons prepared by the preparation method of the present invention. The medium used in the co-culture is not particularly limited, but a medium in which nerve cells can survive is desirable. For example, Neurobasal (registered trademark) medium (Life Technologies), N2 supplement (Thermo Fisher Scientific) or B27 (registered trademark) Supplement with Gibco (registered trademark) B27 (registered trademark) Supplement (Thermo Fisher Scientific) Added DMEM / F12 medium can be used. The culture temperature is preferably 33 to 37 ° C, more preferably 37 ° C. The culture period is not particularly limited, but it can usually be analyzed in about 1 to 2 weeks.

One aspect of the present invention is a method for preparing a neural tissue model, comprising the following steps: (1) introducing at least two types of inducers into human cells; (2) inducing differentiation of the human cells. Obtaining astrocyte-like cells; and (3) co-culturing the astrocyte-like cells and neurons obtained in step (2), wherein the inducer comprises NFIA, and The preparation method includes at least one of NICD and Tet2CD, and the human cell is a human somatic cell (excluding astrocytes), a human pluripotent stem cell or a human adult stem cell.

One aspect of the present invention is a method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, comprising the following steps: (1) a cell derived from a patient with a nervous system disease, or a cell derived from the patient with a nervous system disease A step of preparing an astrocyte-like cell from a cell derived from a healthy person artificially introduced with a mutation that specifically exists in the gene of said gene by the method for preparing an astrocyte-like cell of the present invention; (2) step A step of preparing a neural tissue model by co-culturing astrocyte-like cells obtained in (1) and neurons; (3) contacting the neural tissue model obtained in step (2) with a test substance; And the method comprising the step of evaluating the efficacy or toxicity of the test substance. Another embodiment of the present invention is a method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, comprising the following steps: (1) from the cells derived from a healthy person, the astrocyte-like cells of the present invention. A step of preparing an astrocyte-like cell by the preparation method; (2) a step of knocking out or knocking down a nervous system disease-related gene in the astrocyte-like cell obtained in step (1); (3) step (2) A step of preparing a neural tissue model by co-culturing astrocyte-like cells and neurons obtained in (4); (4) contacting the neural tissue model obtained in step (3) with the test substance, The method comprising the step of assessing the efficacy or toxicity of Although it does not specifically limit as a nervous system disease, For example, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, autism, Alexander disease, Rett syndrome etc. are mentioned.

One aspect of the present invention is an inducer from a human cell to an astrocyte-like cell, comprising at least one inducer or at least one vector comprising a nucleic acid encoding the inducer as a component, The inducer comprises NFIA and comprises at least one of NICD and Tet2CD, and the human cell is a human somatic cell (excluding astrocytes), a human pluripotent stem cell or a human adult stem cell (Hereinafter also referred to as “the inducer of the present invention”). The inducer of the present invention can be used in the method for preparing an astrocyte-like cell of the present invention. For example, by bringing the inducer into contact with a human cell, introducing the inducer or at least one vector containing a nucleic acid encoding the inducer into the cell, and then culturing the cells under appropriate conditions, the cells are astrocytes. Differentiation can be induced into like-like cells.

One aspect of the present invention includes at least two types of inducers, or at least one vector comprising a nucleic acid encoding the inducer; and an instruction manual describing the method of preparing the astrocyte-like cells of the present invention. A kit for inducing human cells to astrocyte-like cells, wherein the inducer comprises NFIA and at least one of NICD and Tet2CD, and the human cells are human somatic cells (astrocytes). And the like, which are human pluripotent stem cells or human adult stem cells (hereinafter also referred to as “kit of the present invention”). The instruction manual in the kit of the present invention may be in a form provided on the web without being included in the kit of the present invention.

The kit of the present invention is selected from an expression inhibitor of at least one gene selected from p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and / or p14ARF, p53, p21, p16INK4a and Rb At least one inhibitor and / or expression enhancer of at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, and / or MDM2, cyclin D1, CDK4, CDK6 And at least one activator selected from E2F. Preferably, an expression inhibitor of at least one gene selected from p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and / or at least one selected from p14ARF, p53, p21, p16INK4a and Rb An inhibitor and / or an expression enhancer of at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene may be further included. More preferably, an expression inhibitor of at least one gene selected from p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and / or at least one selected from p14ARF, p53, p21, p16INK4a and Rb One inhibitor may further be included. More preferably, it may further contain an expression inhibitor of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene. Particularly preferably, it may further comprise an expression inhibitor of at least one gene selected from the p14ARF gene, the p53 gene, the p21 gene and the p16INK4a gene.

The expression inhibitor of p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and the inhibitor of the gene product of these genes are not particularly limited, but may be used to inhibit the expression of the gene. (Eg, nucleic acids such as siRNA, shRNA (for example, RNA encoded by DNA having the nucleotide sequences shown in SEQ ID NOs: 14 to 21) and miRNA) and the gene products used Those listed above (eg, p53 dominant negative mutant, inactive Rb mutant, and pifthrin-α) can be used.

The expression enhancer of MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene and the activator of gene products of these genes are not particularly limited, but can be used to increase the expression of the gene As mentioned above (for example, the expression vectors of these genes, inactive TALEN nuclease, inactive TALEN nuclease and inactive Cas9 nuclease), and those that can activate the gene product Those well known to those skilled in the art can be used.

The present invention will be described more specifically with reference to the following examples and reference examples, but the present invention is not limited thereto.

Example 1 Induction of differentiation from human fibroblasts to astrocyte-like cells (1) Preparation of a lentiviral vector containing a nucleic acid encoding an inducer A lentiviral vector, NICD, containing a nucleic acid encoding NFIA (SEQ ID NO: 1) A lentiviral vector containing a nucleic acid encoding (SEQ ID NO: 2) and a lentiviral vector containing a nucleic acid encoding Tet2CD (SEQ ID NO: 3) were prepared. Specifically, a lentiviral plasmid that expresses an inducer (NFIA, NICD, or Tet2CD) downstream of the CBh promoter was obtained by using pCAG-HIVgp and pCMV-VSV-G-RSV-Rev plasmids (obtained from Dr. Hiroyuki Miyoshi, RIKEN) At the same time, the cells were introduced into 293T cells in Gibco (registered trademark) FreeStyle (trademark) 293 Expression Medium (manufactured by Invitrogen) (hereinafter also simply referred to as “Freestyle (trademark)”). 16-20 hours after introduction, the medium was replaced with fresh medium. After further culturing for 48 hours, the culture supernatant was filtered using a 0.45 μm filter. The resulting supernatant was concentrated or concentrated and then introduced into various cells as a lentiviral vector.

(2) Introduction of lentiviral vector into human fibroblasts and induction of differentiation DMEM medium containing human adult fibroblasts (NHDF-Ad, Lonza) was placed at 5 × 10 ⁴ in a 6-well plate or 10 cm dish. Cells were seeded at a density of cells / well or 3 × 10 ⁵ cells / dish. 24 hours after seeding, the medium was replaced with a medium containing the lentiviral vector prepared in (1) and 4 μg / mL polybrene. 16-20 hours after infection, the medium was replaced with fresh medium. Cells were maintained in media for an additional 48 hours before being grown using AGM media ™ (Lonza). The medium was changed every 3 days until analysis. Analysis was performed 2-3 weeks after infection.

(3) Analysis by immunocytochemistry Analysis of the obtained astrocyte-like cells by immunocytochemistry according to the method described in Kohyama, J. et al., The Journal of cell biology, 2010, 189, 159-170 went. Specifically, the cells were fixed with 4% paraformaldehyde at room temperature. After fixation, the cells were washed with phosphate buffered saline (PBS), and then the cells were washed with a primary antibody-containing 0.3% Triton / 5% FBS / PBS solution (antibody dilution solution) at room temperature for 3 hours or once at 4 degrees. Treated overnight. After the primary antibody treatment, the cells were washed with PBS, and then treated with a diluted antibody solution containing a secondary antibody bound with a fluorescent marker at room temperature for 60 to 90 minutes, followed by washing with PBS. Nuclei were stained with DAPI or Hoechst. Stained cells were mounted on glass slides.

(4) Results When NFIA was introduced into human fibroblasts, GFAP-expressing cells were not observed. When mouse neurofibroblasts were used, differentiation into GFAP-expressing cells could be induced by introducing only NFIA (see Reference Example 1 below).
NFAP is expressed when NFIA and NICD are introduced into human fibroblasts, when NFIA and Tet2CD are introduced into human fibroblasts, and when NFIA, NICD and Tet2CD are introduced into human fibroblasts It was possible to induce differentiation into astrocyte-like cells. In particular, when three inducers were introduced, differentiation into astrocyte-like cells could be induced with a high efficiency of 45.1% (FIG. 1).
It was confirmed that the obtained astrocyte-like cells did not express normally expressed smooth muscle actin (SMA) in the case of fibroblasts.

Example 2
Quantification of neurite length from neurons when astrocyte-like cells and neurons are co-cultured A functional feature of astrocytes is their ability to support neurite growth from neurons. In order to clarify that the astrocyte-like cells obtained in Example 1 have this function, human neonatal dermal fibroblasts (NHDF-Neo) and human adult dermal fibroblasts (NHDF-Ad) (both In the same manner as described in Example 1, the lentiviral vector incorporating the NFIA cDNA (SEQ ID NO: 1) and the lentivirus incorporating the NICD cDNA (SEQ ID NO: 2) were used. Vector and a lentiviral vector incorporating Tet2CD cDNA (SEQ ID NO: 3) were introduced, and astrocyte-like cells induced to differentiate by culturing for 2-3 weeks were Millicell cell culture inserts (24 wells, PCF membrane, 0.4 μm). , Purchased from Millipore) and cultured at 37 ° C. for one day. In addition, E15.5 mouse-derived cerebral cortical neurons were seeded on glass coverslips using DMEM / F12 medium supplemented with Gibco (registered trademark) B27 (registered trademark) Supplement, and cultured at 37 ° C for one day. After the medium in the Millicell cell culture insert seeded with astrocyte-like cells was replaced with B27 / DMEM / F12 medium, the Millicell cell culture insert was set on a coverslip for 3 days and co-cultured at 37 ° C. As a control group, human neonatal fibroblasts (NHDF-Neo), human adult dermal fibroblasts (NHDF-Ad) and human primary cultured astrocytes (HNA) were co-cultured with cerebral cortical neurons derived from E15.5 mice. What was done was used. The neurite length was quantified by labeling neurons with anti-MAP2 antibody (Sigma), then acquiring images with a fluorescence microscope, and using Image J software with neuron J plug-in (Meijering et al., Journal of the International Society for Analytical Cytology, 2004, 58, 167-176). The results are shown in FIG. When astrocyte-like cells and neurons were co-cultured, neurite outgrowth promotion from neurons was significantly observed as compared to the case of co-culture with fibroblasts and neurons as a control group.

Example 3
Measurement of glutamate uptake ability of astrocyte-like cells A functional feature of astrocytes includes the ability to take up glutamate. In order to clarify that the astrocyte-like cells obtained in Example 1 have this function, the following experiment was performed. Coat human adult dermal fibroblasts (NHDF-Ad), astrocyte-like cells obtained by introducing NFIA, NICD and Tet2CD in Example 1 and human primary cultured astrocytes with poly-L-Lysine (Sigma) Seeded in 24-well plates. Various cells were mixed with sodium ion-containing buffer or sodium to which L-glutamic acid (Nacalai) and tritium-labeled L-glutamic acid (L- [2,3,4-3H]) (American Radiolabeled Chemicals Inc.) were added. Incubation was carried out at 37 ° C. using an ion-free buffer. After incubation, the cells were washed with a sodium ion-free buffer and lysed with 0.1N NaOH solution. The cell lysate was mixed with a liquid scintillation cocktail (National Diagnostics), and the radiation dose was measured using a liquid scintillation counter. Moreover, the total protein amount of various cells was quantified by the BCA method. The glutamic acid uptake amount was evaluated by correcting the sodium ion-dependent uptake amount with the total protein amount. The results are shown in FIG.

Example 4 Induction of differentiation from human lt-NES cells to astrocyte-like cells Neural stem cells derived from human ES cells and iPS cells are known to resist differentiation into astrocyte-like cells (Falk, A. et al., PloS ONE, 2012, Vol. 7 (1), e29597). The effect by introducing the inducer used in the present invention was tested on such neural stem cells.

As the starting cells, lt-NES cells derived from human iPS cells (Dr. Austin Smith, obtained from Cambridge University) were used. It-NES cells maintain stable proliferative ability and properties as early neuroepithelial cells in the developing brain. lt-NES cells are known to have weaker glial properties (Falk, A. et al., PloS ONE, 2012, Vol. 7 (1), e29597).

Using a plasmid vector incorporating NFIA cDNA, a plasmid vector incorporating NICD cDNA, and a plasmid vector incorporating Tet2CD cDNA (obtained from Cell Biolabs, Inc), a retroviral vector was prepared as follows. .
The plasmid vector was introduced into Plat-E cells in FreeStyle ™. 16-20 hours after introduction, the medium was replaced with fresh medium. After further culturing for 48 hours, the culture supernatant was filtered using a 0.45 μm filter. The obtained supernatant was used as a retrovirus vector in this example after being concentrated or concentrated.
Lentiviral plasmid (Takahashi, K. et al., Cell, 2007,131 (5): 861-72.) Expressing mouse Slc7a1 downstream of the UbC promoter, FreeStyle together with pCAG-HIVgp and pCMV-VSV-G-RSV-Rev plasmids It was introduced into 293T cells in (trademark), and the prepared lentivirus was introduced into lt-NES cells.
Thereafter, the retroviral vector prepared above was added to the medium containing the lt-NES cells (DMEM / F12 medium containing 1% N2 Supplement and 0.1% B27 Supplement (hereinafter also referred to as “the medium for lt-NES”)). In addition, the cells were cultured at 37 ° C. for 24 hours, and then the medium was changed to an lt-NES medium containing 1% FBS to prepare astrocyte-like cells expressing GFAP. The percentage of GFAP-expressing cells after one week from the introduction of the retroviral vector was more than 20%. Furthermore, after 2 weeks, almost all lt-NES cells were induced into GFAP-expressing cells (FIG. 4).

Example 5 Induction of differentiation from human iPS cells to astrocyte-like cells A culture vessel in which human iPS cells (obtained from iPS Cell Research Institute, Kyoto University) were coated with iMatrix-511 (purchased from Nippi Co., Ltd.), and AK03 Maintenance culture was performed using a medium (purchased from Ajinomoto Co., Inc.). In the same manner as in Example 1 (1), a lentiviral vector containing a nucleic acid encoding an inducer was prepared and introduced into human iPS cells. IPS cells into which the lentiviral vector was introduced were subcultured 72 hours after introduction into a culture vessel not coated with iMatrix-511 using AGM medium. The medium was changed every 3 to 4 days to prepare astrocyte-like cells expressing GFAP from human iPS cells. Note that three types of inducers were used: NFIA, NICD, and Tet2CD. The percentage of GFAP-expressing cells after 2 weeks from the introduction of the vector was about 80% of the total.

Example 6 Preparation of astrocyte-like cells using a viral vector containing all nucleic acids encoding NFIA, NICD and Tet2CD, or an episomal vector (1) Porcine teschovirus-1-derived 2A peptide from Tet2CD, NFIA and NICD Ligation was performed using cDNA. A lentiviral vector incorporating this sequence was introduced into human adult fibroblasts (NHDF-Ad, Lonza) and cultured for 11 days. The obtained cells were GFAP positive. A fluorescence micrograph is shown in FIG.
(2) Tet2CD and NFIA and NICD cDNAs were ligated using porcine teschovirus-1-derived 2A peptide cDNA, and an episomal vector incorporating this sequence was prepared.
Thereafter, the episomal vector prepared above was introduced into the lt-NES cells and cultured for 7 days using an lt-NES medium containing 1% FBS. Thereafter, the cells were further cultured for 7 days using AGM medium. The obtained cells were GFAP positive. A fluorescence micrograph is shown in FIG.

Example 7 Preparation of an in vitro disease model of Alexander disease Alexander disease is known to be a disease caused by GFAP gene mutation (Brenner, M. et al., Nature genetics, 2001, 27, 117-120). In the GFAP gene of Alexander disease patients, arginine 239 of GFAP is mutated to cysteine due to a missense point mutation (C715T) in the gene (R239C). Moreover, it is known that the expression of αB crystallin (CRYAB) is enhanced in astrocytes of Alexander disease patients (Der Perng, M. et al., American journal of human genetics, 2006, 79, 197-213). Moreover, it is known that mutant GFAP proteins aggregate to form Rosenthal fibers in astrocytes of Alexander disease patients (Dinda, AK et al., Acta neuropathologica, 1990, 79, 456-460).

It was tested whether or not the astrocyte-like cells of the present invention can be used in an in vitro disease model of Alexander disease.
Using the CRISPR-Cas9 system (Ran, FA et al., Cell, 2013, 154, 1380-1389), lt-NES cells having a missense point mutation (C715T) in the GFAP gene were prepared. Thereafter, in the same manner as in Example 4, a retroviral vector containing a nucleic acid encoding NFIA, a retroviral vector containing a nucleic acid encoding NICD, and a retroviral vector containing a nucleic acid encoding Tet2CD were introduced into the cells, Astrocyte-like cells expressing GFAP were prepared.

In the obtained astrocyte-like cells, expression of CRYAB was significantly higher than that of the control group (astrocyte-like cells derived from lt-NES cells before introducing the missense point mutation (C715T)). (FIG. 6). Furthermore, rosental fibers were observed in the obtained astrocyte-like cells. The obtained astrocyte-like cells can be used as an in vitro disease model of Alexander disease for elucidation of the molecular mechanism and etiology of the disease, and screening for a therapeutic drug for the disease.

Example 8 Generation of an in vitro disease model of Rett syndrome Rett syndrome is caused by mutations in the MECP2 gene present on the X chromosome. Recent findings on mouse genes suggest that astrocytes play an important role in the development of Rett syndrome (Ballas, N et al., Nature neuroscience, 2009, 12, 311-317; Nguyen, MV et al., The Journal of neuroscience, 2012, 32, 10021-10034). In addition, astrocyte-like cells derived from iPS cells derived from Rett syndrome patients have been reported to adversely affect neuronal maturation (Williams et al., Hum Mol Genet. 2014 Jun 1; 23 (11 ): 2968-80).

It was tested whether the astrocyte-like cells of the present invention can be used in an in vitro disease model of Rett syndrome.
MECP2 protein in astrocyte-like cells derived from human adult fibroblasts prepared by introducing NFIA, NICD and Tet2CD in Example 1 and astrocyte-like cells derived from lt-NES cells prepared in Example 4 In order to suppress the expression of shRNA, shRNA against the target sequence of MECP2 (SEQ ID NO: 24) was expressed by a lentivirus. In addition, a lentivirus expressing a mutant MECP2 that was not suppressed by the shRNA was introduced simultaneously with the shRNA. These cells were cultured for 3 days with cerebral cortical neurons derived from E15.5 mice. The results are shown in FIGS.

Neurons seeded on astrocyte-like cells expressing shRNA against MECP2 were predominantly inhibited in neurite outgrowth compared to the control group. A neural tissue model in which astrocyte-like cells expressing shRNA against MECP2 and neurons are co-cultured is an in vitro disease model of Rett syndrome, for elucidation of the molecular mechanism and etiology of the disease, and screening for a therapeutic drug for the disease Can be used.

Example 9 Effect of p16INK4a in Differentiation Induction from Human Adult Fibroblasts to Astrocyte-Like Cells A lentiviral vector incorporating p16INK4a cDNA (SEQ ID NO: 25) was prepared in the same manner as in Example 1 (1). . In addition, an empty lentiviral vector not incorporating the cDNA was prepared and used as a control.

The prepared lentiviral vector was a lentiviral vector incorporating the NFIA cDNA (SEQ ID NO: 1) prepared in Example 1 (1), a lentiviral vector incorporating the NICD cDNA (SEQ ID NO: 2), and Tet2CD. It was introduced into human adult dermal fibroblasts (NHDF-Ad) (purchased from Lonza Co., Ltd.) together with cDNA (SEQ ID NO: 3) and cultured for 11 days. FIG. 9 shows the ratio between the number of GFAP-positive cells and the number of DAPI-positive cells after culture, and the ratio between the number of GFAP-positive cells after culture and the initial number of cells at the start of culture. When p16INK4a was introduced, the differentiation induction efficiency from human adult fibroblasts to astrocyte-like cells was significantly reduced compared to the control.

Example 10 Effect of knockdown of p16INK4a gene, p53 gene, p14ARF gene and p21 gene in induction of differentiation from human adult fibroblasts to astrocyte-like cells Nucleic acids encoding shRNA against p14ARF gene (SEQ ID NOs: 14 and 15) A lentiviral vector incorporating a nucleic acid encoding a shRNA against the p53 gene (SEQ ID NOS: 16 and 17), a nucleic acid encoding a shRNA against the p21 gene (SEQ ID NO: 18 and 19) A lentiviral vector, a lentiviral vector incorporating a nucleic acid encoding shRNA against the p16INK4a gene (SEQ ID NOs: 20 and 21), and a nucleotide sequence that does not exist in human adult fibroblast genes The nucleic acid (SEQ ID NO: 22 and 23) lentiviral vector (control) incorporating the encoding shRNA to, were prepared in the same manner as in Example 1 (1).

Each of the prepared lentiviral vectors, a lentiviral vector incorporating the NFIA cDNA (SEQ ID NO: 1) prepared in Example 1 (1), a lentiviral vector incorporating the NICD cDNA (SEQ ID NO: 2), and It was introduced into human adult dermal fibroblasts (NHDF-Ad) (purchased from Lonza Co.) together with Tet2CD cDNA (SEQ ID NO: 3) and cultured for 11 days. The ratio between the number of GFAP positive cells and the number of DAPI positive cells after culture, and the ratio between the number of GFAP positive cells after culture and the initial number of cells at the start of culture are shown in FIGS. Compared with the case of introducing the control shRNA, when the shRNA that knocks down the p16INK4a gene, the p53 gene, the p14ARF gene and the p21 gene is introduced, the differentiation induction efficiency from human adult fibroblasts to astrocyte-like cells is improved. Significantly increased.

Reference Example 1 Differentiation induction from mouse fibroblasts to astrocyte-like cells Mouse NFIA, NICD, Tet2CD, and REST / NRSF-VP16 were selected as candidate inducers. Retrovirus vectors containing nucleic acids encoding these inducers (SEQ ID NOs: 1 to 4 respectively) were prepared in the same manner as in Example 4. The retroviral vector was introduced into mouse embryonic fibroblasts (MEF). In the introduction, one of the retroviral vectors was used, or two or three of them were used in combination. GFAP expression was examined 4 days after factor introduction. The ratio of GFAP expressing cells is shown in FIG.

For induction of differentiation from mouse fibroblasts to astrocyte-like cells, NFIA alone was sufficient as an inducer. It was also revealed that the differentiation induction efficiency is increased by combining NFIA and other factors.

The base sequences of the nucleic acids used in the Examples and Reference Examples of the present application, as well as the amino acid sequences of the peptides that can be used in the present invention and the base sequences of the nucleic acids are shown below.

SEQ ID NO: 1: Nucleotide sequence of mouse NFIA cDNA. In the sequence, the underlined portion indicates the HA tag sequence.

SEQ ID NO: 2: Mouse NICD cDNA sequence. In the sequence, the wavy line indicates the Myc tag sequence, and the underline indicates the linker sequence.

SEQ ID NO: 3: base sequence of mouse Tet2CD cDNA. In the sequence, the underlined portion indicates the FLAG tag sequence.

SEQ ID NO: 4: nucleotide sequence of cDNA of mREST / NRSF-VP16. In the sequence, the broken line portion indicates a FLAG tag sequence, the underline portion indicates a sequence encoding mREST / NRSF, the wavy portion indicates a linker sequence, and the italic portion indicates a sequence encoding VP16.

SEQ ID NO: 5: Nucleotide sequence of human NFIA cDNA (NM — 001145511.1).

SEQ ID NO: 6: nucleotide sequence of human NICD cDNA (part of NM — 017617.4).

SEQ ID NO: 7: nucleotide sequence of human Tet2CD cDNA (part of NM_001127208.2).

SEQ ID NO: 8: Amino acid sequence of mouse NFIA (NP_035035.1).

SEQ ID NO: 9: Amino acid sequence of mouse NICD (part of NP_032740.3).

SEQ ID NO: 10: Amino acid sequence of mouse Tet2CD (part of NP_001035490.2).

SEQ ID NO: 11: amino acid sequence of human NFIA (NP_001138983.1).

SEQ ID NO: 12: Amino acid sequence of human NICD (NP_060087.3).

SEQ ID NO: 13: Amino acid sequence of human Tet2CD (NP_001120680.1).

SEQ ID NO: 14: nucleotide sequence of the forward strand of DNA encoding shp14ARF In the sequence, the capital letter indicates the part complementary to the target.

SEQ ID NO: 15: nucleotide sequence of reverse strand of DNA encoding shp14ARF In the sequence, the capital letter indicates the part complementary to the target.

SEQ ID NO: 16: nucleotide sequence of the forward strand of DNA encoding shp53 In the sequence, the capital letter indicates the part complementary to the target.

SEQ ID NO: 17: base sequence of reverse strand of DNA encoding shp53 In the sequence, the capital letter indicates the part complementary to the target.

SEQ ID NO: 18: nucleotide sequence of the forward strand of DNA encoding shp21 In the sequence, the capital letter indicates the part complementary to the target.

SEQ ID NO: 19: base sequence of reverse strand of DNA encoding shp21 In the sequence, the capital letter indicates the part complementary to the target.

SEQ ID NO: 20: nucleotide sequence of the forward strand of DNA encoding shp16INK4a In the sequence, the capital letter indicates the part complementary to the target.

SEQ ID NO: 21: base sequence of reverse strand of DNA encoding shp16INK4a In the sequence, the capital letter indicates the part complementary to the target.

SEQ ID NO: 22: nucleotide sequence of the forward strand of DNA encoding shRNA used as a control in Example 10 In the sequence, the capital letter indicates the part complementary to the target.

SEQ ID NO: 23: base sequence of reverse strand of DNA encoding shRNA used as control in Example 10 In the sequence, the capital letter indicates the part complementary to the target.

SEQ ID NO: 24: Base sequence in MECP2 gene targeted by shRNA used in Example 8.

SEQ ID NO: 25: base sequence of cDNA of p16INK4a (NM_000077.4).

According to the method of the present invention, astrocyte-like cells having characteristics similar to those of astrocytes, which have been difficult to obtain conventionally, can be rapidly and efficiently obtained in large quantities from easily obtainable human cells. In addition, an in vitro disease model can be constructed using the astrocyte-like cells, and can be used for elucidation of the molecular mechanism and pathogenesis of the disease, and screening for a therapeutic drug for the disease.

All publications and patent literature cited herein are hereby incorporated by reference in their entirety. While specific embodiments herein have been described herein for purposes of illustration, it will be appreciated by those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. Will be easily understood.

Claims

A method for preparing in vitro astrocyte-like cells from human cells comprising the following steps:
(1) introducing at least two kinds of inducers into human cells; and (2) obtaining astrocyte-like cells by inducing differentiation of the human cells,
Wherein the inducer comprises NFIA and comprises at least one of NICD and Tet2CD;
The preparation method, wherein the human cells are human somatic cells (excluding astrocytes), human pluripotent stem cells or human adult stem cells.
The preparation method according to claim 1, wherein the inducer includes NFIA, NICD, and Tet2CD.
The preparation method according to claim 1 or 2, wherein the human cells are human fibroblasts.
The preparation method according to claim 1 or 2, wherein the human cell is a human iPS cell or a human neural stem cell.
The human cell is a cell derived from a patient with a nervous system disease, or a cell derived from a healthy person artificially introduced with a mutation that specifically exists in the gene of a cell derived from the patient with a nervous system disease The preparation method according to any one of claims 1 to 4.
The preparation method according to any one of claims 1 to 5, wherein the introduction of the inducer in the step (1) comprises introducing at least one vector containing a nucleic acid encoding the inducer.
In the step (1),
inhibits the expression of at least one gene selected from p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene,
inhibits at least one selected from p14ARF, p53, p21, p16INK4a and Rb,
Increases the expression of at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, or activates at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F The preparation method according to any one of claims 1 to 6, further comprising:
An astrocyte-like cell prepared by the method according to any one of claims 1 to 7.
A method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, comprising the following steps:
(1) From a cell derived from a patient with a nervous system disease, or a cell derived from a healthy person into which a mutation that specifically exists in a gene of a cell derived from a patient with the nervous system disease has been artificially introduced into a gene, A step of preparing an astrocyte-like cell by the method according to any one of the above; and (2) contacting the astrocyte-like cell obtained in step (1) with the test substance to thereby determine the effectiveness of the test substance. Or said method comprising the step of assessing toxicity.
A method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, comprising the following steps:
(1) a step of preparing an astrocyte-like cell from a cell derived from a healthy person by the method according to any one of claims 1 to 7;
(2) a step of knocking out or knocking down a nervous system disease-related gene in the astrocyte-like cell obtained in step (1); and (3) an astrocyte-like cell obtained in step (2) and a test substance The method comprising the steps of: contacting and evaluating the efficacy or toxicity of the test substance.
An in vitro neural tissue model comprising the astrocyte-like cell according to claim 8 and a neuron.
A method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, comprising the following steps:
(1) From a cell derived from a patient with a nervous system disease, or a cell derived from a healthy person into which a mutation that specifically exists in a gene of a cell derived from a patient with the nervous system disease has been artificially introduced into a gene, A step of preparing an astrocyte-like cell by the method according to any one of the above;
(2) a step of preparing a neural tissue model by co-culturing astrocyte-like cells and neurons obtained in step (1);
(3) The said method including the process of contacting the nerve tissue model obtained by process (2), and a test substance, and evaluating the effectiveness or toxicity of a test substance.
A method for evaluating the effectiveness or toxicity of a test substance against a nervous system disease, comprising the following steps:
(1) a step of preparing an astrocyte-like cell from a cell derived from a healthy person by the method according to any one of claims 1 to 7;
(2) a step of knocking out or knocking down a nervous system disease-related gene in the astrocyte-like cell obtained in step (1);
(3) a step of preparing a neural tissue model by co-culturing astrocyte-like cells and neurons obtained in step (2);
(4) The method comprising the step of contacting the nerve tissue model obtained in step (3) with a test substance to evaluate the effectiveness or toxicity of the test substance.
An inducer from a human cell to an astrocyte-like cell, comprising at least one kind of inducer or at least one vector containing a nucleic acid encoding the inducer, wherein the inducer is NFIA And at least one of NICD and Tet2CD, and the human cell is a human somatic cell (excluding astrocytes), a human pluripotent stem cell or a human adult stem cell.
A human cell comprising at least one inducer, or at least one vector comprising a nucleic acid encoding said inducer; and an instruction manual describing the method according to any one of claims 1-7 To astrocyte-like cells, wherein the inducer comprises NFIA and comprises at least one of NICD and Tet2CD, and the human cells are human somatic cells (excluding astrocytes) The kit, which is a human pluripotent stem cell or a human adult stem cell.
an expression inhibitor of at least one gene selected from p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and / or at least one inhibitor selected from p14ARF, p53, p21, p16INK4a and Rb, and And / or expression enhancer of at least one gene selected from MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, and / or at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F The kit of claim 15 further comprising an activator.